PPCNg介导人羊膜间充质干细胞治疗大鼠宫腔粘连的研究

A study of PPCNg-mediated effect of human amniotic mesenchymal stem cells in the treatment of intrauterine adhesion in rats

摘要:

目的：探讨PPCNg聚合物（polyethylene glycol citrate-co-N-isopropylacrylamide gelatin，PPCNg）介导人羊膜间充质干细胞（human amniotic mesenchymal stem cells，hAMSCs）治疗宫腔粘连（intrauterine adhesions，IUA）的可行性和有效性。方法：将PKH26标记的hAMSCs在不同浓度PPCNg中培养，48 h后荧光显微镜下观察细胞状态，CCK-8法分析细胞毒性，评价PPCNg的材料毒性。雌性SD级大鼠随机分为假手术组、单纯IUA模型组（模型组）、PPCNg治疗IUA组（PPCNg组）、hAMSCs治疗IUA组（hAMSCs组）及PPCNg联合hAMSCs治疗IUA组（PPCNg+hAMSCs组）。建立IUA模型2周后，假手术组不处理，其余建模的各组分别宫腔内注射PBS、PPCNg、hAMSCs的PBS悬液及hAMSCs的PPCNg悬液。宫腔注射2周后，行子宫组织石蜡切片HE染色和Masson染色检测子宫内膜腺体及纤维化情况，免疫组化检测子宫内膜中转化生长因子-β1（transforming growth factor-β1，TGF-β1）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、波形蛋白（vimentin，VIM）、α平滑肌肌动蛋白(α-smooth muscle actin，α-SMA）、细胞角蛋白19（cytokeratin 19，CK19）及E-钙粘附蛋白（E-cadherin，E-Ca）表达情况；冰冻切片荧光显微镜下观察子宫内膜中hAMSCs分布情况。结果：①在不同浓度PPCNg中hAMSCs贴壁生长，状态良好；②PPCNg无细胞毒性，材料毒性分级为1级（合格）；③与假手术组相比，模型组腺体数目减少（P=0.000），纤维化面积比增高（P=0.000）；与模型组相比，PPCNg组、hAMSCs组及PPCNg+hAMSCs组的腺体数目不同程度增多（P=0.000），纤维化面积比不同程度减少；④与模型组相比，PPCNg组、hAMSCs组及PPCNg+hAMSCs组TGF-β1、VEGF、VIM及α-SMA的表达均不同程度降低，其中PPCNg+hAMSCs组明显降低（P=0.000），而CK19及E-Ca表达则不同程度升高，其中PPCNg+hAMSCs组明显升高（P=0.000）；⑤PPCNg+hAMSCs组PKH26标记的hAMSCs远远多于hAMSCs组。结论：PPCNg无细胞毒性，能显著提高hAMSCs治疗IUA的有效性。

Abstract:

Objective：To investigate polyethylene glycol citrate-co-N -isopropylacrylamide gelatin（PPCNg）-mediated feasibility and effectiveness of human amniotic mesenchymal stem cells（hAMSCs） in the treatment of intrauterine adhesion（IUA）. Methods：The PKH26-labeled hAMSCs were cultured in different concentrations of PPCNg and were observed for cell status with a fluorescence microscope after 48 hours. CCK-8 assay was used to evaluate the cytotoxicity and material toxicity of PPCNg. The female Sprague-Dawley rats were randomly divided into sham operation group，IUA group（model group），PPCNg-treated IUA group（PPCNg group），hAMSCs-treated IUA group（hAMSCs group），and PPCNg+hAMSCs-treated IUA group（PPCNg+hAMSCs group）；two weeks after IUA modeling，the above five groups except the sham operation group（left untreated） were injected with PBS，PBS-suspended PPCNg，PBS-suspended hAMSCs，and PBS-suspended hAMSCs and PPCNg solution，respectively，in the uterine cavity. Two weeks after the intrauterine injection，paraffin sections of the uterine tissue were treated with HE staining and Masson staining to measure the number of endometrial glands and fibrosis status. Immunohisto－chemistry was used to determine the expression of transforming growth factor-β1（TGF-β1），vascular endothelial growth factor （VEGF），vimentin（VIM），α-smooth muscle actin（α-SMA），cytokeratin 19（CK19），and E-cadherin（E-Ca） in the endometrium. The distribution of hAMSCs in the endometrium was evaluated with a fluorescence microscope using frozen sections. Results：In different concentrations of PPCNg，hAMSCs adherently grew well，and PPCNg was not cytotoxic with its material toxicity classified as grade 1（qualified）. Compared with the sham operation group，the model group had a significantly reduced number of glands and a significantly increased fibrosis area ratio（both P=0.000）. Compared with the model group，the PPCNg group，the hAMSCs group，and the PPCNg+hAMSCs group had different degrees of increase in the number of glands（P=0.000） and different degrees of decrease in the fibrosis area ratio；meanwhile，the three groups had different degrees of decrease in the expression of TGF-β1，VEGF，VIM，and α-SMA（with a significant decrease observed in the PPCNg+hAMSCs group[P=0.000]） and different degrees of increase in the expression of CK19 and E-Ca（with a significant increase observed in the PPCNg+hAMSCs group[P=0.000]）. The PPCNg+hAMSCs group had more PKH26-labeled hAMSCs than the hAMSCs group. Conclusion：PPCNg has no cytotoxicity and can significantly improve the effectiveness of hAMSCs in the treatment of IUA.